The overall goal of this K08 Mentored Clinical Scientist Career Development Award is to provide me with the mentored training to become an independent investigator as a physician-scientist pursuing translational neuroscience research in psychostimulant abuse. My specific career goals include application of genetic, behavioral and imaging tools towards development of an improved understanding of the basic neurobiology and synaptic signaling mechanisms underlying drug abuse and addiction. To accomplish this, I propose to use the fly Drosophila melanogaster as a model system to identify relevant molecular targets efficiently and for eventual testing and validation in rodent models. The fly provides a powerful model system to study mechanisms of psychostimulant signaling given its advantage of allowing concurrent investigation of biochemical pathways at molecular and behavioral levels. In addressing gaps in my training, my K08 training goals are: 1) to develop expertise in fly neurobiology and genetics, and 2) to develop expertise in imaging pre- and postsynaptic dopamine neuronal signaling. As proposed for this award, I will use a multidisciplinary approach combining behavioral and imaging studies to investigate the molecular pathophysiology underlying psychostimulant abuse, with a focus on amphetamine (AMPH) action. Though the primary sites of action for AMPH have been identified, the downstream signaling pathways are poorly understood. My colleagues and I have shown that fly larvae respond to AMPH by crawling faster and this is dependent both on presynaptic dopamine transporter and postsynaptic dopamine (DA) D2 receptors (D2R). While postsynaptic D2R signals may be mediated by G1i/o-dependent and/or arrestin-dependent/G1i/o-independent (Arr-dependent) downstream signaling pathways, it is not known whether AMPH action is dependent on one or both pathways. Importantly, the kinase Akt behaves as an intermediary between several signaling molecules downstream of D2R known to mediate AMPH-stimulated locomotion and understanding its regulation may shed light on molecular mechanisms of AMPH action. I will test the hypothesis that AMPH stimulation of DA postsynaptic neurons in the fly leads to D2R-dependent activation of both G1i/o-dependent and Arr-dependent signaling pathways. I will address 2 specific aims in this work: 1) to test whether AMPH-stimulated locomotion is dependent on the Arr-dependent and/or G1i/o-dependent pathways using RNAi knockdown of signaling molecules in both pathways, and 2) to determine in vivo effects on Akt activity of AMPH-mediated postsynaptic D2R activation via multiphoton imaging of the Akt biosensor within the intact living fly brain. These novel approaches and findings will facilitate further characterization of AMPH's molecular actions and move us toward critically needed treatments and to better models of stimulant pathophysiology. PUBLIC HEALTH RELEVANCE: Abuse of psychostimulant drugs such as amphetamine represents a major public health problem with a broad array of adverse health consequences, including dependence, overdose and death. Although these drugs have been some of the most used and abused substances for the last century, our knowledge of their molecular mechanisms of action in the brain remains rudimentary. This Mentored Clinical Scientist Research Career Development Award (K08) will enable Dr. Zachary Freyberg to elucidate mechanisms of action for stimulants such as amphetamine and therefore open the door to novel therapeutic interventions.